Tetanus is caused by tetanus neurotoxin (TeNT), which initially enters motor neurons and then traffics retrogradely into the spinal cord. It is then released from motor neurons, re-enters inhibitory neurons and blocks inhibitory input onto motor neurons, causing involuntary muscle contraction.Thisisinasharpcontrasttobotulinumneurotoxins(BoNTs),whichactwithinmotor neurons to cause the disease botulism. TeNT and BoNTs share the same overall structure/function, yet their distinct trafficking pathways in neurons produce two different diseases. While the pathology is well established, the molecular mechanism underlying the retrogradetransportofTeNTremainselusive. The classic view is that TeNT and BoNTs recognize distinct receptors, and TeNT?s unique receptor mediates its retrograde transport. Recent evidence and our preliminary studies demonstratethatthecell-surfacereceptorisnotinvolvedinthesortingprocess,andtheregion beyondthereceptor-bindingdomainofTeNTisrequiredforsorting.Indeed,thecrystalstructure of TeNT reveals that its protease domain, translocation domain, and receptor-binding domain formuniqueinterfacesatendosomalpHlevels.Thus,weproposeamajorrevisionoftheclassic view: specific interactions among different domains of TeNT are critical for its efficient sorting into the retrograde transport pathway. Here we will test our hypothesis by designing specific mutations that disrupt inter-domain interactions in TeNT and evaluate the activity of these mutanttoxinsonmousemodelsinvivo.Wealsoseektoidentifythekeyhostsortingfactorsthat mediateTeNTretrogradesortingthroughunbiasedglycanarrayscreensandaffinitypurification. Successful completion of our aims will establish a new paradigm in understanding TeNT retrograde transport, and may provide key insights into endosomal sorting and retrograde transport mechanisms in general. Such an understanding will also lay the foundation for developingeffectivetoxin-basedtoolstodelivertherapeuticsintothecentralnervoussystem.